Erythrocyte sodium buffering capacity status correlates with self-reported salt intake in a population from Livingstone, Zambia

Background Salt impairs endothelial function and increases arterial stiffness independent of blood pressure. The mechanisms are unknown. Recent evidence suggests that there is a possible link between salt consumption and sodium buffering capacity and cardiovascular disease but there is limited evidence in the populations living in Sub-Saharan Africa. The aim of our study was to explore the relationship between erythrocyte sodium buffering capacity and sociodemographic, clinical factors, and self-reported salt consumption at Livingstone Central Hospital. Methods We conducted a cross sectional study at Livingstone Central hospital among 242 volunteers accessing routine medical checkups. Sociodemographic and dietary characteristics were obtained along with clinical measurements to evaluate their health status. Sodium buffering capacity was estimated by erythrocyte sodium sensitivity (ESS) test. We used descriptive and inferential statistics to describe and examine associations between erythrocyte sodium sensitivity and independent variables. Results The median age (interquartile range) of the study sample was 27 (22, 42) years. 54% (n = 202) and 46% (n = 169) were males and females, respectively. The majority (n = 150, 62%) had an ESS of >120%. High salt intake correlated positively with ESS or negatively with vascular sodium buffering capacity. Conclusions Self-reported high salt intake was associated with poor vascular sodium buffering capacity or high ESS in the majority of middle-aged Zambians living in Livingstone. The poor vascular sodium buffering capacity implies a damaged vascular glycocalyx which may potentially lead to a leakage of sodium into the interstitium. This alone is a risk factor for the future development of hypertension and cardiovascular disease. However, future studies need to validate vascular function status when using ESS testing by including established vascular function assessments to determine its pathophysiological and clinical implications.


th December, 2021 To the Editor in Chief
Dear Editor,

Ref: Response to reviewer comments
The above matter refers.
We thank the reviewers for their constructive and positive review of our manuscript. We revised the manuscript in accordance with the various review comments (indicated in bold) and hope that it is now acceptable for publication.
Responses to the reviewers Reviewer #1: Masenga and colleagues want to define what factors are related to sodium buffering capacity in a population living in Zambia. To establish sodium buffering capacity, they have used the erythrocyte sodium sensitivity test as developed by Oberleithner a couple of year ago. The idea that sodium can be stored in the endothelial glycocalyx and the glycocalyx of the erythrocyte may have great impact on the current concepts of sodium homeostasis and pathophysiology of salt sensitivity. As such, this paper addresses an important topic, but I have some important concerns.

Major comments -Abstract
In the introduction the authors state that high sodium consumption leads to reduced sodium buffering capacity. I don't think this statement is completely true. Data from animals and intervention studies indicate that high sodium effects can be neutralized by increasing buffering capacity, for instance, in the skin (Machnik A, Nature). Glycocalyx dimension might change after an IV sodium load, but necessarily after high sodium intake (Rorije NM, Anesthesiology) We agree with the reviewer that this statement is misleading. We have corrected it to imply only that there is a possible relationship between salt intake and sodium buffering capacity and CVD which is supported by literature. We appreciate this comment.
Results. Line 39. The word 'about' should be removed. Please report exact numbers in the result section.

We have removed as suggested. Thank you
Results. Line 41. The expression 'erythrocyte sodium sensitive' is an unclear expression and seems more an interpretation of the results than a true finding.

P.O Box 60009, Livingstone Campus.
Email:smasenga@mu.ac.zm website:www.mu.ac.zm Contacts: +260 966674774 Thank you for the suggestion. We have modified appropriately and reported only the finding.

Methods section
The description of the included patients is very unclear. It seems a pretty random selection of health care workers and patients visiting the hospital on a regular base.
We agree very much with the reviewer that the population under study was not described clearly. We have clarified this by adding some descriptive details. The population consisted of health care workers attending to routine medical checkups. We excluded any participants who were ill at the time of study.
The way sodium consumption has been estimated is not clearly described. For instance, what does an adapted version of the food frequency questionnaire exactly mean?
We have now elaborated in detail how salt consumption was assessed in the structured questionnaire. In addition, as requested by another reviewer, we have additionally sampled a quarter of the total participants and estimated their dietary salt intake using 24-hour urine samples to validate perceived salt intake. We found a positive correlation and our data is further supported by prior research.
The ESS test that has been used for estimating sodium buffering capacity has in my view never been validated. Sodium buffering capacity can be demonstrated after tissue ashing methods or by MRI techniques. Unfortunately, no data with regard to these accepted methods are available to my understanding.

We have removed 'about' as suggested. Thank you.
Line 125 reads a bit difficult.

P.O Box 60009, Livingstone Campus.
Email:smasenga@mu.ac.zm website:www.mu.ac.zm Contacts: +260 966674774 We have re-written this sentence to read clearly. Thank you. Table 2 shows that red blood cell indices (RBC, Hb, and Ht) differ for each ESS category. Given the lack of validation of the ESS test, I wonder whether ESS is not merely another red blood cell marker than a reflection sodium buffering in glycocalyx. It seems that the results of the multiple linear analysis do not take away this concern.
Much appreciated for this suggestion. Our understanding of ESS test is that since the buffering capacity of red blood cells mirror that of the glycocalyx, a reduction in RBC count would mean a reduction in the buffering of sodium. This is expected as shown in the linear regression results and table 2. But truly, this does not take away the possibility that ESS may just be another red blood cell marker, a concern raised by the reviewer. However, we have now provided supplementary data (S2 table) that was based on additional analysis where we have included estimated salt intake in the multiple linear analysis and this takes away the concern of the red cell indices; they did not remain significantly associated with ESS when actual estimated salt intake was included in the model. Thank you for this suggestion.

Figure 1 A and B show the regression between categorical variables. This is not very informative.
Thank you for the suggestion. We have removed the two figures on gender nd employment Reviewer #2: Masenga performed a very interesting study showing a correlation between the erythrocyte glycocalyx, ABI and self-reported salt consumption in a healthy cohort. This is of great importance and can be useful as low-cost, fast and simple tool for the prevention of hypertension and cardiovascular events which are often triggered by high salt consumption, especially in salt-sensitive individuals. However, I have some concerns:

A clear description of the methods is missing and should be provided
We have expanded the methods section to accurately elaborate the methods to ensure that they are reproducible. We appreciate the concern and fill that the methods section is now clearly described. We are happy to receive further specific advice if necessary.
2. Also, a clear structure of the manuscript is missing (e.g. figure legends appear in the discussion section)